Effect of Epichloë Endophyte on the Growth and Carbon Allocation of Its Host Plant Stipa purpurea under Hemiparasitic Root Stress

Epichloë endophytes not only affect the growth and resistance of their host plants but also confer nutrient benefits to parasitized hosts. In this study, we used Pedicularis kansuensis to parasitize Stipa purpurea, both with and without endophytic fungi, and to establish a parasitic system. In this study, endophytic fungal infection was found to increase the dry weight of the leaf, stem, and leaf sheath, as well as the plant height, root length, tiller number, aboveground biomass, and underground biomass of S. purpurea under root hemiparasitic stress. Meanwhile, the 13C allocation of the leaf sheaths and roots of S. purpurea increased as the density of P. kansuensis increased, while the 13C allocation of the leaf sheaths and roots of E+ S. purpurea was lower than that of E− S. purpurea. The 13C allocation of the stem, leaf sheath, and root of E+ S. purpurea was higher than that of its E− counterpart. Furthermore, the content of photosynthetic 13C and the 13C partition rate of the stems, leaves, roots, and entire plant of S. purpurea and P. kansuensis transferred from S. purpurea increased as the density of P. kansuensis increased. These results will generate new insights into the potential role of symbiotic microorganisms in regulating the interaction between root hemiparasites and their hosts.


Introduction
Epichloë endophytes commonly grow in the apoplast of the aerial tissues of a variety of cool-season grasses without causing disease [1,2].Following an extended period of adaptive evolution, a mutually beneficial symbiotic relationship is established between endophytic fungi and cold season grasses.In this relationship, endophytes are capable of acquiring nutrients from their host plants and utilizing the host plant as their habitat sites [3].Endophytic fungi have been found to influence plant productivity, plant-plant interactions, the structure and biodiversity of plant communities, and the conservation and restoration of ecosystems [4,5].Thus far, the importance of endophytic fungal communities in mitigating key abiotic stresses has been well studied.However, there are few reports on whether grasses and their symbiotic microorganisms can offset the harmful effects of root hemiparasitic plants in the broader context of biological stress.
Root hemiparasitic plants depend largely on haustoria to obtain nutrients and water from their host plants [6,7] and subsequently weaken their growth [8,9].Hemiparasitic plants compete with host plants for light resources, which in turn alters the competitive relationship between host and non-host plants.This competition has a significant impact on the composition and structure of plant communities [10,11].At present, the majority of root hemiparasitic plants have become obnoxious forbs that seriously harm agriculture and animal husbandry [12,13].For example, Striga hermonthica is parasitic on sorghum [14], maize [15], and other crops, resulting in an estimated yield reduction of 65% or even no harvest in some cases.In China, there are many kinds of root hemiparasitic plants, but most are scattered and rarely clustered [16], which has resulted in insufficient attention being paid to the hazardous risks of this group.However, in recent years, some root hemiparasitic plant species have shown significant spreading trends, which pose a potential threat to the sustainable use of grasslands and the healthy development of animal husbandry [17,18].A typical example is the expanded population of P. kansuensis into Qinghai, Xinjiang, southwestern Gansu, western Sichuan, and western Tibet [19].
P. kansuensis is an endemic annual or biennial root hemiparasitic herb found in China [20].Since 2000, the native species of P. kansuensis has been expanding rapidly, which has threatened the local livestock industry [17,21].The hemiparasitic nature of its roots is an exclusive survival strategy employed by P. kansuensis.Specifically, it relies on its specialized structure called haustoria to obtain a portion of water and nutrients from gramineous and leguminous plants [22], resulting in the biomass of gramineous and leguminous plants rapidly decreasing [20,[23][24][25].Recent studies have demonstrated that plant symbiotic microorganisms have the ability to greatly enhance their host's tolerance to root parasitic stress [26,27].In a study conducted by Sui et al. [28], it was discovered that the growth performance of both hosts and parasites in Trifolium repens and Pedicularis rex/tricolor legume pairs was greatly enhanced through inoculation with Glomus mosseae.This inoculation also helped alleviate the harm caused by P. rex/tricolor.At present, most studies focus on Epichloë endophytes in improving host grass resistance ability [29].However, there are currently few reports on the regulation of carbon uptake and allocation by endophytic fungi in grasses and root hemiparasitic systems.
Our previous study found that P. kansuensis could establish root parasitic relationships with 18 sympatric plants and that S. purpurea was revealed to be the main host of P. kansuensis [30].Meanwhile, the infection rate of endophytic fungi in the degraded grassland of S. purpurea was found to be more than 90%.The type of endophytic fungi responsible for the infection was identified as Epichloë inebrians [31].Therefore, in this study, we utilized endophyte-infected (E+) and endophyte-free (E−) S. purpurea as our materials to study the plant growth of S. purpurea under different intensities of P. kansuensis parasitization and normal growth conditions.Accordingly, this study aims to address the following questions: (1) What are the growth characteristics of E+ and E− S. purpurea and P. kansuensis? (2) What are the total photosynthetic carbon and distribution characteristics of S. purpurea when transferred to P. kansuensis? (3) How does photosynthetic carbon sequestration and allocation differ between E+ and E− S. purpurea?

Experimental Materials
In October 2019, in the natural grassland of Ganzihe Town, Haiyan County, Haibei Prefecture, Qinghai Province (37 • 07 09 N, 100 • 38 42 E), the mature seeds of S. purpurea and P. kansuensis were harvested.The harvest site is 3370 m above sea level, which belongs to the classic alpine grassland, the grassland group species is S. purpurea, and the associated species are Agropyron cristatum and Poa pratensis.In our previous study [31,32], we identified the tested material, so we are very sure that it is the seeds of S. purpurea and P. kansuensis.According to the detection method of endophytic fungi in grass by Li et al. [33], the endophyte infection status of S. purpurea was determined by the microscopic examination of leaf sheath pieces and seeds stained with aniline blue [34]; the endophytic fungal carrier rate of S. purpurea was detected and found to be as high as 90%.At the same time, 20 seeds of S. purpurea were selected for seed skin disinfection and then placed on Solanum tuberosum glucose agar medium for 28 days at room temperature and under dark conditions.Referring to the identification method of endophytic fungi in S. purpurea, Bao et al. [31] amplified the endophytic fungal sequences, and phylogenetic trees were constructed using Tub, Tef, and Actin specific primers.The endophytic fungus infected with S. purpurea was identified as Epichloë inebrians.S. purpurea E+ seeds were subjected to a soaking process in 70% Topsin-M for eight hours, followed by washing with distilled water to remove any remaining fungicide and to obtain E− seeds.
The experiment was conducted in December 2021 at the smart greenhouse of the Academy of Animal Science and Veterinary Medicine at Qinghai University.E+ and E− seeds of a uniform and complete size were selected and disinfected with 1% sodium hypochlorite for 10 min.The seeds were then rinsed six times with deionized water and blotted with sterilized filter paper to remove any remaining water on the seed surface.Subsequently, the seeds were sown into polyethylene plastic pots measuring 10 cm in diameter and 15 cm in height.To eliminate the influence of nutrients and microorganisms, sand with a diameter of 1-2 mm was used as the growth substrate.The sand had undergone three rounds of rinsing in deionized water and was autoclaved at 120 • C twice for two hours each.After a period of six weeks, the endophytic fungal status of the E+ seedlings was determined to be 100%, while that of the E-seedlings was 0%, according to the method described by Bao et al. [31].As shown in Figure 1a,b, the arrows refer to the hyphae of E. inebrians, and Figure 1c shows that E+ seeds were subjected to a soaking process in 70% Topsin-M for eight hours, followed by washing with distilled water to remove any remaining fungicide and to obtain E− seeds, so its seedings did not have hyphae.tuberosum glucose agar medium for 28 days at room temperature and under dark conditions.Referring to the identification method of endophytic fungi in S. purpurea, Bao et al. [31] amplified the endophytic fungal sequences, and phylogenetic trees were constructed using Tub, Tef, and Actin specific primers.The endophytic fungus infected with S. purpurea was identified as Epichloë inebrians.S. purpurea E+ seeds were subjected to a soaking process in 70% Topsin-M for eight hours, followed by washing with distilled water to remove any remaining fungicide and to obtain E− seeds.
The experiment was conducted in December 2021 at the smart greenhouse of the Academy of Animal Science and Veterinary Medicine at Qinghai University.E+ and E− seeds of a uniform and complete size were selected and disinfected with 1% sodium hypochlorite for 10 min.The seeds were then rinsed six times with deionized water and blotted with sterilized filter paper to remove any remaining water on the seed surface.Subsequently, the seeds were sown into polyethylene plastic pots measuring 10 cm in diameter and 15 cm in height.To eliminate the influence of nutrients and microorganisms, sand with a diameter of 1-2 mm was used as the growth substrate.The sand had undergone three rounds of rinsing in deionized water and was autoclaved at 120 °C twice for two hours each.After a period of six weeks, the endophytic fungal status of the E+ seedlings was determined to be 100%, while that of the E-seedlings was 0%, according to the method described by Bao et al. [31].As shown in Figure 1a,b, the arrows refer to the hyphae of E. inebrians, and Figure 1c shows that E+ seeds were subjected to a soaking process in 70% Topsin-M for eight hours, followed by washing with distilled water to remove any remaining fungicide and to obtain E− seeds, so its seedings did not have hyphae.

Experimental Design and 13 C Labeling
Within three months of establishing the parasitic relationship between P. kansuensis and the E+ and E− type S. purpurea plants, a total of six pots were randomly selected from eight treatments.Three pots from each treatment were then placed in a sealed 13 CO 2 gas isotope labeling dish with a 60 L internal volume to conduct labeling.The remaining three pots, which were labeled with 13 C isotopes, were used as the control group.A CO 2 temperature and gas monitor (AZ7752) was positioned inside the isotope labeling chamber to monitor the concentration of CO 2 in real time.Additionally, a small fan (N15, 2.5 W) was installed within the chamber to facilitate the mixing of the air and the 13 CO 2 gas.To ensure a stable temperature, the outer compartment of the marker box was filled with running water, which ensured that the temperature of the marker box remained between 24 • C and 32 • C. A rubber stopper was attached to the inlet of the isotope-labeling chamber.The labeling gas was obtained from the 13 CO 2 bag using a 50 mL syringe and injected into the labeling chamber (Wuhan Estop Technology Co., Ltd., Wuhan, China, purity ≥ 99.8%, abundance ≥ 99%) at a slow pace.Figure 2 shows a scheme of the isotopic labeling box.
a stable temperature, the outer compartment of the marker box was filled with running water, which ensured that the temperature of the marker box remained between 24 °C and 32 °C.A rubber stopper was attached to the inlet of the isotope-labeling chamber.The labeling gas was obtained from the 13 CO2 bag using a 50 mL syringe and injected into the labeling chamber (Wuhan Estop Technology Co., Ltd., Wuhan, China, purity ≥ 99.8%, abundance ≥ 99%) at a slow pace.Figure 2 shows a scheme of the isotopic labeling box.
The 13 CO2 labeling assay was initiated on 7 July 2022, and labeling was performed daily from 09:00 to 15:00 for three consecutive days.After 3 days of labelling, the samples were taken.The 13 C-labeled plant was placed in the labeling chamber.The labeling gas from the 13 CO2 canister was introduced once the CO2 concentration in the canister dropped to 200 ppm.After introducing 35 mL of 13 CO2 gas, the CO2 concentration inside the box was monitored until it dropped to 250-300 ppm.At this point, it was essential to reintroduce another 35 mL of 13 CO2 gas to maintain a stable CO2 concentration between 400 and 500 ppm inside the marker box.Additionally, after the third introduction of 35 mL of 13 CO2 gas, regular CO2 gas could be introduced to maintain consistent levels of total 13 CO2.To ensure that plant photosynthesis was not affected by the CO2 concentration, a No. 8 selfsealing bag (18 × 25 cm) was utilized to cover the P. kansuensis in the S. purpurea-P.kansuensis root parasitic system.Additionally, tin foil was wrapped around the outside of the bag to prevent photosynthesis by P. kansuensis, thus eliminating its potential impact on the assay results.Samples of S. purpurea and P. kansuensis were collected from the treatment and control groups less than one hour after the completion of isotope labeling.The samples were collected from the root, stem, leaf, and leaf sheath of S. purpurea and from the root, stem, and leaf of P. kansuensis.The 13 CO 2 labeling assay was initiated on 7 July 2022, and labeling was performed daily from 09:00 to 15:00 for three consecutive days.After 3 days of labelling, the samples were taken.The 13 C-labeled plant was placed in the labeling chamber.The labeling gas from the 13 CO 2 canister was introduced once the CO 2 concentration in the canister dropped to 200 ppm.After introducing 35 mL of 13 CO 2 gas, the CO 2 concentration inside the box was monitored until it dropped to 250-300 ppm.At this point, it was essential to reintroduce another 35 mL of 13 CO 2 gas to maintain a stable CO 2 concentration between 400 and 500 ppm inside the marker box.Additionally, after the third introduction of 35 mL of 13 CO 2 gas, regular CO 2 gas could be introduced to maintain consistent levels of total 13 CO 2 .To ensure that plant photosynthesis was not affected by the CO 2 concentration, a No. 8 self-sealing bag (18 × 25 cm) was utilized to cover the P. kansuensis in the S. purpurea-P.kansuensis root parasitic system.Additionally, tin foil was wrapped around the outside of the bag to prevent photosynthesis by P. kansuensis, thus eliminating its potential impact on the assay results.Samples of S. purpurea and P. kansuensis were collected from the treatment and control groups less than one hour after the completion of isotope labeling.The samples were collected from the root, stem, leaf, and leaf sheath of S. purpurea and from the root, stem, and leaf of P. kansuensis.

Plant Harvest and Analysis
After undergoing multiple washes with deionized water, the samples were subjected to a temperature of 105 • C for a duration of 30 min and subsequently dried in an oven at 65 • C until they reached a constant weight.The samples were then ground and passed through an 80-mesh sieve.The total carbon content and 13 C content of the collected samples were analyzed using a Vibratome grinding machine (GT200, Beijing Grademan Instruments Co., Ltd., Beijing, China).
The total carbon content and 13 C abundance were measured using a SerCon Integra 2 (Suzhou Elam Analytical Instruments Co., LTD., Suzhou, China) fully automated stable isotope mass spectrometry system.Both the treatment and control samples were weighed to 0.3 mg (with an accuracy of 0.01 mg) and then packed into tin boats or tin capsules.After placing the sample in the inlet tray, it was combusted in a combustion tube containing silver wire, copper oxide, and chromium oxide at a temperature of 1000 • C. To reduce the amount of gas obtained, a reduction tube filled with copper wire and heated to 600 • C was used.The sample was separated into a column at a temperature of 60 • C after removing the water.An elemental analyzer detector was then used to calculate its carbon content by comparing it to a standard sample.The concentrations and abundances of carbon isotopes were directly measured using a stable isotope mass spectrometry system [36].

13 C Calculations
The total carbon content of each organ in the sample, C i (mg) was calculated by multiplying the carbon mass fraction C i (%) of each organ with the biomass C b (g) of that fraction [37]: The 13 C atomic excess percentage of each organ atom% 13 C i excess (i.e., the 13 C abundance of each organ in the treated group samples (atom 13 C i %) minus the background value of 13 C abundance of each organ in the control group samples (atom 13 C o %)) was calculated as follows: To determine the amount of 13 C photosynthesis in each organ in the sample, we used Equation (3) by substituting the 13 C atomic percentage.The variable 13 C i represents the amount of 13 C photosynthesis in each organ in the sample (mg), and C i represents the quantity of carbon in each organ (mg): 13 The fractional 13 C allocation rate Pi% could be calculated using Equation (4), where 13 C i represents the amount of 13 C photosynthesis in each organ in the sample (mg), and Σ 13 C i represents the total amount of 13 C photosynthesis in each organ of the plant sampled (mg):

Data Analysis
All statistical analyses were conducted using IBM SPSS Statistics Version 26.0.Before performing the analyses, the data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using the Brown-Forsythe test.Statistical analyses for determining significant differences in observations were applied throughout the study using two-way ANOVA, one-way ANOVA, or Student's t-tests.The significance level was set at p < 0.05 for all statistical analyses.

Effects of Endophytic Fungal Infection and P. kansuensis Parasitism on S. purpurea and P. kansuensis Growth Characteristics
Under root hemiparasitic stress, there was a significant effect of endophytic fungal infection on the dry weight of the leaf, stem, leaf sheath, tiller number, and aboveground biomass and underground biomass (Table 1, p < 0.01).Meanwhile, the parasitic density of P. kansuensis had a significant effect on the dry weight of the leaf, stem, and leaf sheath, as well as the plant height, root length, tiller number, aboveground biomass, and underground biomass of S. purpurea (Table 1, p < 0.01).Apart from the plant height and tiller number, the interaction between the endophytic fungal infection and P. kansuensis parasitic density had a significant effect on the rest of the growth characteristics of S. purpurea (Table 1, p < 0.01).As the parasitic density of P. kansuensis increased, apart from the dry weight of the leaf sheath, the dry weight of the leaf, stem, aboveground biomass, underground biomass, plant height, root length, and tiller number of S. purpurea exhibited a downward trend (Figure 3a-h).When S. purpurea was parasitized by P. kansuensis, the dry weight of the leaf, stem, and leaf sheath, as well as the tiller number, aboveground biomass, and underground biomass of E+ S. purpurea, were significantly higher than those of E− plants (Figure 3a-h, p < 0.05).Without the parasitic effect of P. kansuensis on S. purpurea, the dry weight of the leaf and stem, as well as the root length, tiller number, aboveground biomass, and underground biomass, were the highest compared to those of samples receiving other treatments (Figure 3a-h).
Table 1.Two-way ANOVA results for the effect of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the biomass (including the dry weight of the leaf sheath, stem, and leave), plant height, root length, and tiller number of S. purpurea.The parasitic density had a significant effect on the aboveground and underground biomass of P. kansuensis (Table 2, p < 0.01).However, it did not have an effect on the plant height, root length, or underground biomass (Table 2, p > 0.05).At the same time, the endophytic fungal infection status of S. purpurea had significant effects on the root length, as well as the aboveground and underground biomasses of P. kansuensis (Table 2, p < 0.05).However, it did not have an effect on the plant height (Table 2, p > 0.05).In addition, the interaction between endophytic fungal infection and P. kansuensis parasitic density had a significant effect on the underground biomass of P. kansuensis, but there was no impact on the plant height, root length, and aboveground biomass (Table 2, p < 0.01).As the parasitic density of P. kansuensis increased, there was a noticeable declining trend in plant height, but there was a noticeable increasing trend in both the aboveground and underground biomasses of P. kansuensis (Figure 4a-d).Furthermore, when there were no host plants, the plant height, root length, and aboveground and underground biomasses of P. kansuensis increased with the increase in plant density (Figure 4a-d).Additionally, for P. kansuensis of parasitized E+ S. purpurea, the plant height and root length were lower than those of parasitized E− plants (Figure 4a,b), and the aboveground and underground biomasses were higher than those of parasitized E− plants (Figure 4a,b).The maximum number of P. kansuensis haustoria per unit area in the root of S. purpurea was the highest (i.e., 0.36 cm 2 ) when there was no endophytic fungal infection and only one parasite of P. kansuensis (Figure 5).Conversely, the lowest maximum number of P. kansuensis haustoria per unit area in the root of S. purpurea was 0.12 cm 2 , which occurred when there was both endophytic fungal infection and one parasite of P. kansuensis (Figure 5).As the density of P. kansuensis parasitica increased, the haustoria number of P. kansuensis parasitica per unit area in the root area of E+ S. purpurea increased, while this haustoria number decreased in the root area of E− S. purpurea, although the differences were not significant (Figure 5, p > 0.05).The maximum number of P. kansuensis haustoria per unit area in the root of S. purpurea was the highest (i.e., 0.36 cm 2 ) when there was no endophytic fungal infection and only one parasite of P. kansuensis (Figure 5).Conversely, the lowest maximum number of P. kansuensis haustoria per unit area in the root of S. purpurea was 0.12 cm 2 , which occurred when there was both endophytic fungal infection and one parasite of P. kansuensis (Figure 5).As the density of P. kansuensis parasitica increased, the haustoria number of P. kansuensis parasitica per unit area in the root area of E+ S. purpurea increased, while this haustoria number decreased in the root area of E− S. purpurea, although the differences were not significant (Figure 5, p > 0.05).3.2.Effects of Endophyte Fungal Infection and P. kansuensis Parasitica on the Total Carbon Content of S. purpurea and P. kansuensis

Plant Growth
Endophytic fungal infection and P. kansuensis parasitica had a significant impact on the total carbon content of the leaf sheath, stem, leaves, and roots of S. purpurea under root hemiparasitic stress (Table 3, p < 0.01).Additionally, there was a significant interaction between endophytic fungus infection and P. kansuensis parasitica density that affected the total carbon content of the leaf sheath, stem, leaves, and roots of S. purpura (Table 3, p < 0.01).With the increase in the parasitic density of P. kansuensis, the total carbon content of the stems, leaves, and roots decreased, but this was not the case with the leaf sheath (Figure 6a-d).Additionally, the total carbon contents of the leaf sheath, stem, leaves, and roots of E+ S. purpura were significantly higher than those of the E− plants, regardless of whether the plant was parasitic or not (Figure 6a-d, p < 0.05).
Table 3. Two-way ANOVA results for the effect of the E endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the total carbon content of the leaves, stems, leaf sheaths, and roots of S. purpurea.The endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis were significantly affected by the total carbon content in the stems, leaves, and roots of P. kansuensis (Table 4, p < 0.01).Specifically, the total carbon content of the stems, leaves, and roots of P. kansuensis was significantly influenced by the interaction between the endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis (Table 4, p < 0.05).The total carbon contents of the stems, leaves, and roots of P. kansuensis increased as the density of P. kansuensis increased, irrespective of the presence of its host S. purpurea (Figure 7a,b).At the same time, the total carbon contents of the stems, leaves, and roots of the P. kansuensis parasiticum E+ S. purpurea were higher than those of the P. kansuensis parasiticum E− S. purpurea (Figure 7a,b), but the total carbon contents of the stems, leaves, and roots of the P. kansuensis parasiticum E− P. kansuensis were lower than those of P. kansuensis without a host (Figure 7a,b).The endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis were significantly affected by the total carbon content in the stems, leaves, and roots of P. kansuensis (Table 4, p < 0.01).Specifically, the total carbon content of the stems, leaves, and roots of P. kansuensis was significantly influenced by the interaction between the endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis (Table 4, p < 0.05).The total carbon contents of the stems, leaves, and roots of P. kansuensis increased as the density of P. kansuensis increased, irrespective of the presence of its host S. purpurea (Figure 7a,b).At the same time, the total carbon contents of the stems, leaves, and roots of the P. kansuensis parasiticum E+ S. purpurea were higher than those of the P. kansuensis parasiticum E− S. purpurea (Figure 7a,b), but the total carbon contents of the stems, leaves, and roots of the P. kansuensis parasiticum E− P. kansuensis were lower than those of P. kansuensis without a host (Figure 7a,b).Under root hemiparasitic stress, both endophytic fungal infection and the parasitic density of P. kansuensis had significant impacts on the leaf sheath, stem, leaves, roots, and overall photosynthetic 13 C content (Table 5, p < 0.01).Specifically, the interaction between endophytic fungal infection and P. kansuensis parasitica had a significant impact on the photosynthetic 13 C content in the leaf sheath, leaf, stem, and root of S. purpurea (Table 5, p < 0.05).With the increasing parasitic density of P. kansuensis, the photosynthetic 13 C content in the leaves, stems, and overall structures of E+ and E− S. purpurea showed a decreasing trend, while the same content of the leaf sheath and root showed no significant change (Figure 8a-e).The photosynthetic 13 C contents in the leaf sheath, stem, leaves, and overall structure of E+ S. purpurea were significantly higher compared to the corresponding values for E− plants (Figure 8a-e, p < 0.05).
Table 5. Two-way ANOVA results of the effect of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the photosynthetic 13 C content of the leaves, stems, leaf sheaths, and roots of S. purpurea.The parasitic density and endophytic fungal infection status had significant effects on the 13 C content of the stems, leaves, and overall photosynthesis transferred to S. purpurea and P. kansuensis (Table 6, p < 0.01), and the parasitic density had significant effects on the 13 C content of roots photosynthesis transferred to S. purpurea and P. kansuensis (Table 6, p < 0.01).However, there was no effect on the photosynthetic 13 C content of P. kansuensis that was transferred from the roots of S. purpurea (Table 6, p = 0.61).At the same time, the interaction of the endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis had a significant effect on the photosynthetic 13 C content of P. kansuensis that was transferred from the stems and leaves of S. purpurea (Table 6, p < 0.01).However, it was observed that the photosynthetic 13 C content of the roots and overall structure of P. kansuensis did not show any significant changes when transferred from the stems and leaves of S. purpurea (Table 6, p > 0.05).However, as the parasitic density of P. kansuensis increased, there was a continuous increase in the 13 C content of the stem leaves, roots, and overall photosynthesis of P. kansuensis transferred from S. purpurea (Figure 9a-c).The 13 C content in the stems, leaves, roots, and overall photosynthetic structure of P. kansuensis that was transferred from E+ S. purpurea was higher than that of E− S. purpurea (Figure 9a-c

Effects of Endophyte Fungal Infection and P. kansuensis Parasitica on the Photosynthetic 13 C Allocation Rate of S. purpurea and P. kansuensis
The endophytic fungal infection and the density of P. kansuensis parasites had a significant impact on the allocation of photosynthetic 13 C in the leaves, stems, leaf sheaths, and roots of S. purpurea when subjected to root hemiparasitic stress (Table 7, p < 0.05).Among these components, the interaction between endophytic fungal infection and the parasitic density of P. kansuensis had a significant effect on the photosynthetic 13 C allocation rate in the leaves, stems, leaf sheath, and roots of S. purpurea (Table 7, p < 0.01).As the parasitic density of P. kansuensis increased, the photosynthetic 13 C allocation rates of E+ and E− S. purpurea in the leaf sheath and roots exhibited an upward trend.However, there was no significant change in the photosynthetic 13 C content in the leaves and stems (Figure 10a-d).Under the condition of P. kansuensis parasitism, the photosynthetic 13 C allocation kansuensis density and endophyte status on the stem leaves photosynthetic 13 C content of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the roots photosynthetic 13 C content of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the total photosynthetic 13 C content of S. purpurea.

Effects of Endophyte Fungal Infection and P. kansuensis Parasitica on the Photosynthetic 13 C Allocation Rate of S. purpurea and P. kansuensis
The endophytic fungal infection and the density of P. kansuensis parasites had a significant impact on the allocation of photosynthetic 13 C in the leaves, stems, leaf sheaths, and roots of S. purpurea when subjected to root hemiparasitic stress (Table 7, p < 0.05).Among these components, the interaction between endophytic fungal infection and the parasitic density of P. kansuensis had a significant effect on the photosynthetic 13 C allocation rate in the leaves, stems, leaf sheath, and roots of S. purpurea (Table 7, p < 0.01).As the parasitic density of P. kansuensis increased, the photosynthetic 13 C allocation rates of E+ and E− S. purpurea in the leaf sheath and roots exhibited an upward trend.However, there was no significant change in the photosynthetic 13 C content in the leaves and stems (Figure 10a-d).Under the condition of P. kansuensis parasitism, the photosynthetic 13 C allocation rate of E+ S. purpurea leaves was lower than that of E− S. purpurea.However, the rate of photosynthetic 13 C allocation in E+ S. purpurea stems, leaf sheaths, and roots was higher than that of E− S. purpurea (Figure 10a-d).Under the condition of three-parasitic P. kansuensis, the photosynthetic 13 C allocation rates of the leaves, leaf sheath, and roots of E+ S. purpurea were higher than those of E− S. purpurea, while the photosynthetic 13 C allocation rate of the stems of E + S. purpurea was lower than that of E− S. purpurea (Figure 10a-d).
Table 7. Two-way ANOVA results for the effect of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the 13 C allocation rate of the leaves, stems, leaf sheaths, and roots of S. purpurea.The endophytic fungal infection and the parasitic density of P. kansuensis had significant effects on the 13 C carbon allocation rate of the stem leaves and roots of P. kansuensis (Table 8, p < 0.01).Among these effects, the endophytic fungal infection status of S. purpurea and the parasitic density of P. kansuensis had a significant interaction effect on the 13 C allocation rate of the stem leaves and roots of P. kansuensis (Table 8, p < 0.01).As the parasitic density of P. kansuensis increased, the 13 C allocation rate of the stem leaves of P. kansuensis continued to decrease, and the 13 C allocation rate of the roots of P. kansuensis continued to increase (Figure 11a,b).At the same time, the 13 C allocation rate of P. kansuensis stem leaves parasitic upon E+ S. purpurea was higher than that of E− plants (Figure 11a, p < 0.05).However, when the parasitic density was one, the 13 C allocation rate of P. kansuensis roots parasitic upon E+ S. purpurea was lower than that of E− plants (Figure 11b, p < 0.05).continued to increase (Figure 11a,b).At the same time, the 13 C allocation rate of P. kansuensis stem leaves parasitic upon E+ S. purpurea was higher than that of E− plants (Figure 11a, p < 0.05).However, when the parasitic density was one, the 13 C allocation rate of P. kansuensis roots parasitic upon E+ S. purpurea was lower than that of E− plants (Figure 11b, p < 0.05).

Discussion
Root parasite plants usually rob their hosts of water, carbohydrates, and nutrients to meet their own growth needs [7,38].Numerous studies have demonstrated that root parasitic plants can negatively impact the growth characteristics of their host plants [39,40].In our experiment, we observed that when P. kansuensis parasitized S. purpurea, there was a decrease in the leaf weight, stem weight, sheath weight, aboveground biomass, under-

Discussion
Root parasite plants rob their hosts of water, carbohydrates, and nutrients to meet their own growth needs [7,38].Numerous studies have demonstrated that root parasitic plants can negatively impact the growth characteristics of their host plants [39,40].In our experiment, we observed that when P. kansuensis parasitized S. purpurea, there was a decrease in the leaf weight, stem weight, sheath weight, aboveground biomass, underground biomass, plant height, root length, and tillering number of S. purpurea (Figure 3a-h).Meanwhile, as the parasitic density of P. kansuensis increased, the inhibitory effect also increased gradually.
Some studies have suggested that endophytic fungi can enhance a plant's ability to withstand both biotic and abiotic stresses [41][42][43].In the context of root parasitic stress, our study revealed that E+ S. purpurea exhibited a greater leaf weight, stem weight, sheath weight, aboveground biomass, underground biomass, root length, and tillering number compared to E− plants (Figure 3a-h).In conclusion, we found that endophytic fungal infection can enhance the tolerance of S. purpurea to the root hemiparasitic stress caused by P. kansuensis.Furthermore, when P. kansuensis parasitized S. purpurea with an endophytic fungal infection, it had varying effects on the plant's height, root length, aboveground biomass, and underground biomass.
It is worth noting that after P. kansuensis parasitized E+ S. purpurea, we observed that its stems and leaves easily withered, but new stems and leaves could be regrown.This could be due to the infection of endophytic fungi inhibiting P. kansuensis from getting nutrients from S. purpurea, thereby causing the living environment of P. kansuensis to deteriorate.However, in the case of P. kansuensis parasitized with E+ S. purpurea, its aboveground and underground biomasses were higher than those of P. kansuensis parasitized with E− S. purpurea (Figure 4c,d); the reason for this difference may be that the endophytic fungus infection may enhance the photosynthetic capacity of S. purpurea under stressful conditions.This can lead to the accumulation of more carbohydrates, which would enable S. purpurea and root parasitic plants to meet their growth requirements.Consequently, endophytic fungi help alleviate the root hemiparasitic damage caused by P. kansuensis on S. purpurea.
In this study, we found that when the parasitic density of P. kansuensis was varied, the aboveground, underground, and total biomasses of the E+ S. purpurea and P. kansuensis parasitism systems were higher compared to the E− S. purpurea and P. kansuensis parasitism systems.The results of this study suggest that endophytic fungi enhance the light capture capacity of the aboveground part of S. purpurea by influencing its growth characteristics.Additionally, endophytic fungi promote root growth and improve a plant's ability to compete for nutrient absorption in the subsurface.By reducing the number of haustoria of P. kansuensis per unit area, the nutrient plunder from S. purpurea is minimized, resulting in an increased accumulation of dry matter in the host-parasitic plant system.
The process of plant growth is influenced by both material accumulation and physiological metabolism, which are, in turn, affected by plant photosynthesis.Approximately 90% of the materials required for plant growth are derived from organic compounds produced through photosynthesis, which typically have a carbon content ranging from 40 to 50% [44].Photosynthetic carbon is the starting point of carbon metabolism in plants, which is the most important metabolic activity in the process of plant growth and development.This function mainly includes the assimilation of inorganic carbon (CO 2 ) into organic carbon and the metabolic processes of carbohydrate transformation, transport, accumulation, and decomposition among different plant tissues [45,46].Thus, the quantitative allocation and fixation of photosynthetic carbon is of great significance for studying the allocation of fixed carbon in plants, while also serving as an important means by which to analyze the influence of external factors on plant growth [46,47].
Parasitic plants use root haustorium to establish a parasitic relationship with the host, and part of the carbohydrate produced by the host through photosynthesis is ingested by parasitic plants through phloem channels [7,48].Studies have shown that root hemiparasitic plants can directly inhibit the photosynthetic metabolism of hosts and reduce their aboveground biomass [49][50][51][52].In this study, with the increase in the parasitic density of P. kansuensis, the total carbon contents of the leaves, stems, and roots of S. purpurea decreased, and the total carbon contents of E+ S. purpurea leaves, stems, leaf sheaths, and roots were higher than those of E− plants (Figure 6a-d).This is consistent with Suryanarayanan et al.'s conclusion that endophytic fungi have a positive effect on the photosynthetic characteristics of plants [53].In addition, with the increase in the density of P. kansuensis, the total carbon content of the stem leaves and roots of P. kansuensis increased, regardless of whether there was a host, and the total carbon contents of the stem leaves and roots of P. kansuensis parasitizing E+ S. purpurea were higher than those of P. kansuensis parasitica E− plants.However, the total carbon contents of the stem leaves and roots of P. kansuensis parasitica E− S. purpurea were lower than those of plants without a parasite (Figure 8a,b).On the one hand, the inhibition of photosynthesis in E− S. purpurea under root hemiparasitic stress could have led to a decrease in the carbon allocation absorbed by P. kansuensis.On the other hand, in the pot experiment, both S. purpurea and P. kansuensis may have competed for nutrients, resulting in insufficient nutrient availability for both species.In contrast, the host-free P. kansuensis could fully absorb the required nutrients.
Up to now, the positive effect of endophytic fungi on photosynthetic carbon fixation has been widely accepted [53], but its mechanism needs to be further explored. 13C pulse labeling can be used to study the distribution of photosynthetic carbon in plants at a certain period in time [47,54,55].Therefore, 13 C pulse labeling can be used to study the photosynthetic carbon allocation in the parasitic system established by host-parasite plants [37].In this study, 13 CO 2 isotope labeling was used to quantitatively analyze the photosynthetic 13 C amount and 13 C allocation rate of P. kansuensis that was transferred from S. purpurea and the total carbon content of P. kansuensis.In our experiment, the stem photosynthetic 13C of high-density parasitic E+ S. purpurea was significantly higher compared to that of non-parasitic E+ plants (Figure 8b, p < 0.05), while that of low-density parasitic E+ S. purpurea was higher than that of non-parasitic E+ S. purpurea, although the differences were not significant (Figure 8b, p > 0.05).This may be the result of the transition from a mutualistic relationship to a competitive one between endophytic fungi and S. purpurea.Some studies have shown that endophytic fungi are most frequently distributed in the stem internode and leaf sheath [56], and the difference in the photosynthetic 13 C in the stem and leaf sheath of E+ S. purpurea under different parasitic densities may be the result of different distribution densities of endophytic fungi.At the same time, compared with the high-density parasitism in P. kansuensis, carbon fixed by photosynthesis and carbohydrates formed by carbon cycling can meet the growth requirements of S. purpurea, endophytic fungi, and root parasitism plants.However, in our study, the carbon and carbohydrate synthesized from the stems and leaves of S. purpurea could not meet the above three requirements at the same time under the high-density parasitic stress of P. kansuensis.To maintain its normal growth, S. purpurea preferentially delivers more carbon and carbohydrates to the roots, which is consistent with the result that the root biomass of E+ S. purpurea was higher than that of E− S. purpurea under heavy root parasitic stress.This conclusion is also consistent with the survival strategy that plants preferentially distribute nutrients to the roots, through which they also obtain nutrient resources in the soil, under the condition of nutrient scarcity [57].
It Is worth noting that under conditions of heavy parasitism, the 13 C allocation rates of the stem and root of E+ S. purpurea were lower than those of E− S. purpurea (Figure 10a-d, p > 0.05), although the differences were not significant.However, the photosynthetic 13 C amounts in the stem and root of E+ S. purpurea were significantly higher compared to those of E− plants (Figure 8a-d, p < 0.05).These results indicate that the carbon metabolic efficiency of E+ S. purpurea is higher than that of E− plants under the condition of heavy parasitism of P. kansuensis, which supports the idea that endophytic fungi can improve the photosynthetic capacity of host grasses and thus enhance the carbon utilization efficiency.Meanwhile, the photosynthetic 13 C amounts in the stems, leaves, and whole plants of P. kansuensis that were transferred from E+ S. purpurea were higher than those transferred from E− S. purpurea, but there was no difference in the photosynthetic 13 C amounts of P. kansuensis transferred from the roots (Figure 9a-c).Furthermore, the photosynthetic 13 C allocation rate of P. kansuensis transferred from S. purpurea further supports the above view.Compared with P. kansuensis, which was parasitic upon E− S. purpurea, the photosynthetic allocation rate of 13 C in the stem leaves of P. kansuensis parasitized by E+ S. purpurea was higher, but the photosynthetic allocation rate of 13 C was significantly decreased when the roots were transferred from S. purpurea (Figure 11a,b).These results further indicate that endophytic fungi regulate the distribution ratio of photosynthetic 13 C in the roots and stem leaves of P. kansuensis when transferred from S. purpurea in an environment of heavy parasitism.In other words, P. kansuensis's stem leaves receive a preferential distribution of photosynthetic 13 C, which makes the root's primary source of carbon photosynthetic carbon, which is then used to offset the plant's excessive uptake of S. purpurea's carbohydrates.At the same time, the regulatory mechanism of photosynthetic carbon allocation by endophytic fungi on the transfer of P. kansuensis from S. purpurea could further explain why P. kansuensis accumulated more biomass when parasitized by E+ S. purpurea (Figure 4).

Conclusions
Our study shows that parasitism by P. kansuensis can inhibit the growth of S. purpurea, and the inhibitory effect increases with an increase in the parasitic density of P. kansuensis.Compared with the E− S. purpurea, the plant height, root length, and tiller number of the infected S. purpurea were significantly increased.The results indicate that endophytic fungal infection can increase the total carbon content of S. purpurea under root hemiparasitic stress.Meanwhile, the total carbon contents of stems, leaves, leaf sheathes, and roots were significantly inhibited by P. kansuensis parasitism, and this effect worsened with the increase in the parasitic density of P. kansuensis.However, endophytic fungal infection was able to increase the total carbon content of S. purpurea under parasitic stress.Under the heavy parasitism of P. kansuensis, the carbon metabolic efficiency of E+ S. purpurea was higher than that of E− plants.Finally, endophytic fungi regulate the distribution ratio of photosynthetic 13 C in the roots and stem leaves of P. kansuensis transferred from S. purpurea, with a preference for distribution to the stem leaves of P. kansuensis.This study focused solely on the growth and carbon distribution of S. purpurea under the root parasitism stress caused by endophytic fungi; further research is needed to investigate the allocation of other elements such as nitrogen and phosphorus.

Figure 1 .
Figure 1.Image of endophytic fungal hyphae of S. purpurea leaf sheath.Note: (a) is a hyphae image of the microscopic examination of the S. purpurea leaf sheath collected from natural grassland; (b) is a hyphae image of the microscopic examination of the seedlings' leaf sheath of endophyte infection S. purpurea(E+) used in the experiments; (c) is an image of the microscopic examination of the seedlings' leaf sheath of endophyte none-infection S. purpurea(E−) used in the experiments.

Figure 1 .
Figure 1.Image of endophytic fungal hyphae of S. purpurea leaf sheath.Note: (a) is a hyphae image of the microscopic examination of the S. purpurea leaf sheath collected from natural grassland; (b) is a hyphae image of the microscopic examination of the seedlings' leaf sheath of endophyte infection S. purpurea(E+) used in the experiments; (c) is an image of the microscopic examination of the seedlings' leaf sheath of endophyte none-infection S. purpurea(E−) used in the experiments.

Figure 2 .
Figure 2. Schematic diagram of 13 C−CO 2 isotope labeling for root parasitized systems between S. purpurea and P. kansuensis.

Figure 4 .
Figure 4. Effects of the P. kansuensis density and endophyte status of S. purpurea on the growth of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05); different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of the P. kansuensis density and endophyte status of S. purpurea on the plant height of P. kansuensis; (b) Effects of the P. kansuensis density and endophyte status of S. purpurea on the root length of P. kansuensis; (c) Effects of the P. kansuensis density and endophyte status of S. purpurea on the aboveground biomass of P. kansuensis; (d) Effects of the P. kansuensis density and endophyte status of S. purpurea on the underground biomass of P. kansuensis.

Figure 4 . 22 Figure 5 .
Figure 4. Effects of the P. kansuensis density and endophyte status of S. purpurea on the growth of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05); different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of the P. kansuensis density and endophyte status of S. purpurea on the plant height of P. kansuensis; (b) Effects of the P. kansuensis density and endophyte status of S. purpurea on the root length of P. kansuensis; (c) Effects of the P. kansuensis density and endophyte status of S. purpurea on the aboveground biomass of P. kansuensis; (d) Effects of the P. kansuensis density and endophyte status of S. purpurea on the underground biomass of P. kansuensis.Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 22

Figure 5 .
Figure 5.Effect of S. purpurea endophyte status on the number of haustoria per unit area of parasitic systems of S. purpurea and P. kansuensis.Note: The number of P. kansuensis haustoria is based on the

Figure 6 .
Figure 6.Effects of P. kansuensis density and endophyte status on the total carbon content of leaves, stems, leaf sheaths, and roots of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05).Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the total carbon content of leaves of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the the total carbon content of stems of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the the total carbon content of leaf sheaths of S. purpurea; (d) Effects of P. kansuensis density and endophyte status on the total carbon content of roots of S. purpurea.

Figure 6 .
Figure 6.Effects of P. kansuensis density and endophyte status on the total carbon content of leaves, stems, leaf sheaths, and roots of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05).Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the total carbon content of leaves of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the the total carbon content of stems of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the the total carbon content of leaf sheaths of S. purpurea; (d) Effects of P. kansuensis density and endophyte status on the total carbon content of roots of S. purpurea.

Figure 7 .
Figure 7. Effects of P. kansuensis density and endophyte status on the total carbon content of the stems, leaves, and roots of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).Different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of P. kansuensis density and endophyte status on the total carbon content of leaves of P. kansuensis; (b) Effects of P. kansuensis density and endophyte status on the the total carbon content of stems of P. kansuensis.

Figure 7 .
Figure 7. Effects of P. kansuensis density and endophyte status on the total carbon content of the stems, leaves, and roots of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate

Figure 8 .Figure 8 .
Figure 8. Effects of P. kansuensis density and endophyte status on the leaves, stems, sheath, root, and total photosynthetic 13 C content of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05).Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the leaves photosynthetic 13 C content of S. purpurea; (b) Effects of P. kansuensis density andFigure 8. Effects of P. kansuensis density and endophyte status on the leaves, stems, sheath, root, and total photosynthetic 13 C content of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05).Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the leaves photosynthetic13 C content of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the stems photosynthetic13 C content of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the sheath photosynthetic13 C content of S. purpurea; (d) Effects of P. kansuensis density and endophyte status on the root photosynthetic13 C content of S. purpurea; (e) Effects of P. kansuensis density and endophyte status on the total photosynthetic13 C content of S. purpurea.

Figure 9 .
Figure 9. Effects of the P. kansuensis density and endophyte status on the13 C content of the stem leaves, roots, and total plant of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05); Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05); (a) Effects of P. kansuensis density and endophyte status on the stem leaves photosynthetic13 C content of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the roots photosynthetic13 C content of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the total photosynthetic13 C content of S. purpurea.

Figure 9 .
Figure 9. Effects of the P. kansuensis density and endophyte status on the 13 C content of the stem leaves, roots, and total plant of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05); Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05); (a) Effects of P.kansuensis density and endophyte status on the stem leaves photosynthetic13 C content of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the roots photosynthetic13 C content of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the total photosynthetic13 C content of S. purpurea.

Figure 10 .
Figure 10.Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the leaves, stems, leaf sheaths, and roots of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05); Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the leaves of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the stems of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the leaf sheaths of S. purpurea; (d) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of roots of S. purpurea.

Figure 10 .
Figure 10.Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the leaves, stems, leaf sheaths, and roots of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05); Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate

Figure 11 .
Figure 11.Effects of the P. kansuensis density and endophyte status of S. purpurea on the 13 C allocation rate of the stem leaves, and roots of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).Different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the stem leaves of P. kansuensis; (b) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the roots of P. kansuensis.

Figure 11 .
Figure 11.Effects of the P. kansuensis density and endophyte status of S. purpurea on the 13 C allocation rate of the stem leaves, and roots of P. kansuensis.Note: Data are the mean standard errors.Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).Different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the stem leaves of P. kansuensis; (b) Effects of P. kansuensis density and endophyte status on the 13 C allocation rate of the roots of P. kansuensis.
Different lowercase letters indicate significant differences between different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).One hemiparasite indicates that one S. purpurea plant was parasitized by one P. kansuensis, three hemiparasites indicate that one S. purpurea plant was parasitized by three P. kansuensis, and "without parasite" indicates S. purpurea growing alone; (a) Effects of P. kansuensis density and endophyte status on the dry weight of leaf of S. purpurea; (b) Effects of P. kansuensis density and endophyte status on the dry weight of stem of S. purpurea; (c) Effects of P. kansuensis density and endophyte status on the dry weight of leaf sheath of S. purpurea; (d) Effects of P. kansuensis density and endophyte status on the plant height of S. purpurea; (e) Effects of P. kansuensis density and endophyte status on the root length of S. purpurea; (f) Effects of P. kansuensis density and endophyte status on the tiller number of S. purpurea; (g) Effects of P. kansuensis density and endophyte status on the aboveground biomass of S. purpurea; (h) Effects of P. kansuensis density and endophyte status on the underground biomass of S. purpurea.
Figure 3. Effects of P. kansuensis density and endophyte status on the growth of S. purpurea.Note: Data are the mean standard errors.Different capital letters indicate significant differences between different S. purpurea endophyte statuses at the same P. kansuensis density (p < 0.05).

Table 2 .
Two-way ANOVA results of the effects of the P. kansuensis parasitic density (P) and endophyte fungal infection status of S. purpurea (E) on the plant height, root length, aboveground biomass, and underground biomass of P. kansuensis.

Table 4 .
Two-way ANOVA results of the effects of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the total carbon content of the stems, leaves, and roots of the P. kansuensis.

Table 4 .
Two -way ANOVA results of the effects of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the total carbon content of the stems, leaves, and roots of the P. kansuensis.

Treatments df Total Carbon Content of Stems and Leaves Total Carbon Content of Roots
Different capital letters indicate significant differences between the different P. kansuensis densities in the same S. purpurea endophyte state (p < 0.05).Different lowercase letters indicate significant differences between the different S. purpurea endophyte states at the same P. kansuensis density (p < 0.05).NH indicates that P. kansuensis was not grown with S. purpurea; (a) Effects of P. kansuensis density and endophyte status on the total carbon content of leaves of P. kansuensis; (b) Effects of P. kansuensis density and endophyte status on the the total carbon content of stems of P. kansuensis.3.3.Effects of Endophyte Fungal Infection and P. kansuensis Parasitica on the Photosynthetic 13 C Content of S. purpurea and P. kansuensis ).

Table 6 .
Results of the two-way ANOVA showing the effects of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the13C content of the stem leaves, roots, and total plant of the P. kansuensis.

Table 7 .
Two-way ANOVA results for the effect of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the13C allocation rate of the leaves, stems, leaf sheaths, and roots of S. purpurea.

Table 8 .
Results of the two-way ANOVA showing the effects of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the13C allocation rate of the stem leaf and root of the P. kansuensis.

Table 8 .
Results of the two-way ANOVA showing the effects of the endophyte fungal infection status (E) and parasitic density of the P. kansuensis (P) on the13C allocation rate of the stem leaf and root of the P. kansuensis.